Eph receptors represent the largest subfamily of receptor tyrosine kinases. The Ephs and their ligands, the ephrins, are predominantly expressed in the developing and adult nervous and vascular systems, playing important roles in axon guidance and cell migration. Eph receptors are unique among other receptor kinases in that they fall into two subclasses with distinct ligand specificities, and in that they can also function as ligands activating bi-directional signaling. Understanding the mechanism of Eph/ephrin signaling and its role during development requires a detailed structural and biophysical characterization of the Eph receptors, the ephrins, and their interaction. Our studies of the extracellular functional domains of these molecules during the past five years revealed unique features not previously seen in any other receptor-ligand families and explained many of their unique biochemical and signaling properties. Our future research will continue to integrate the tools of X-ray crystallography, protein biochemistry, protein biophysics, and molecular biology into a program of detailed analysis of the Eph/ephrin recognition and signaling. By studying the three-dimensional structures of the full ectodomains of Ephs, ephrins and of the complete transmembrane molecules, as well as of their various biologically important complexes, we will gain further insight into the unique structural features of this signaling system that have placed it in the center of the development and function of the nervous system. Parallel to our crystallographic experiments will be our biophysical studies of the kinetics and thermodynamics of the ligand/receptor interactions. The combined information will provide invaluable insights into the molecular mechanisms underlying the biological functions of Ephs and ephrins The specific aims of our proposed investigations are: 1. Structural characterization of full Eph ectodomains alone and in complex with ephrins; 2. Structural and biophysical characterization of the Eph/ephrin/ADAM interactions and cleavage; 3. Production, structural and functional characterization of full-length (transmembrane) Eph receptors. PUBLIC HEALTH RELEVANCE: Eph receptors represent the largest subfamily of receptor tyrosine kinases. The Ephs and their ligands, the Ephrins, are predominantly expressed in the developing and adult nervous and vascular systems, playing important roles in axon guidance and cell migration. Eph receptors are unique among other receptor kinases in that they fall into two subclasses with distinct ligand specificities, and in that they can also function as ligands activating bi-directional signaling. Understanding the mechanism of Eph/ephrin signaling and its role during development requires a detailed structural and biophysical characterization of the Eph receptors, the ephrins, and their interaction. We use X-ray crystallography combined with other biophysical techniques to study how these molecules recognize and interact with each other. The specific aims of our proposed investigations are: 1. Structural characterization of full Eph ectodomains alone and in complex with ephrins; 2. Structural and biophysical characterization of the Eph/ephrin/ADAM interactions and cleavage; 3. Production, structural and functional characterization of full-length (transmembrane) Eph receptors.